WO2015058321A1 - Avicennia marina monooxygenase cmo and coding gene and use thereof - Google Patents

Abstract

Disclosed are a monooxygenase CMO derived from Avicennia marina and a coding gene thereof, and a use of the coding gene in the cultivation of transgenic plants having improved salt tolerance and drought tolerance.

Description

 Albino monooxygenase CMO and its coding gene and application

FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and more particularly to a monooxygenase CMO derived from Avicennia marina and its coding gene, and in the cultivation of transgenic plants having improved salt tolerance and drought tolerance Applications. BACKGROUND OF THE INVENTION Drought limits the cultivation of crops by more than 40% of the Earth's land area and poses a serious threat to global agricultural production and food supply. Drought is one of the main environmental constraints on crop yields.

The world's saline-alkali soil is large, about 400 million hectares, accounting for one-third of irrigated farmland. In semi-arid and arid regions with dry climates, salt accumulation continues due to low rainfall and rapid evaporation; salt content in the coastal areas is increased due to seawater intrusion. China's saline-alkali soil is mainly distributed in the northwest, north China, northeast and coastal areas. With the increasing area of greenhouses and the age of cultivation, soil salinization is becoming more and more serious. For most crops, excess Na ^{+ in the} soil can have a toxic effect on the normal growth and metabolism of the plant. Therefore, how to increase crop yield under saline environment has become a very important issue in agricultural production in China.

 It is simple and feasible to use traditional methods to breed salt-tolerant and drought-tolerant plant varieties, but the progress is slow and the limitations are large. With the development of molecular biology technology, a large number of genes related to salt tolerance and drought resistance of plants have been cloned one after another, and plant transgenic technology has made a major breakthrough. This is to effectively use dry land and saline-alkali land to increase crop yield and control salinization and desertification land. Provide new ideas and methods.

The stress resistance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules. When a plant is stressed, it will respond accordingly to reduce or eliminate the damage caused to the plant. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These genes and their expression products can be divided into three categories: (1) genes involved in signal cascade amplification systems and transcriptional control and their expression products; (2) genes that directly contribute to the protection of biofilms and proteins and their expression products; (3) Genes and proteins associated with the uptake and transport of water and ions. Scientists from all over the world have done a lot of work and made breakthroughs in this process (Park S. 2005. Up-regulation of a H ⁺ -pyrophosphatase (H ⁺ -PPase) as a strategy to engineer drought-resistant crop plants. Proc. Natl. Acad. Sci. USA. 102: 18830-18835; ABE H.2003.A rabid op sis AtMYC2 (bHLH) and AtM YB 2 (M YB ) function as transcripicalal activato rs in abscisic acid Signaling. Plant Cell, 15 : 63-78; Zhang ZL. 201 1. Arabidopsis Floral Initiator SKB 1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing through Altering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cell, 23 : 396-41 1). Through biotechnology, the research on stress-tolerant crops, xerophytes and halophytes has achieved remarkable results, and the stress-related genes and signal transduction systems have been further understood.

 However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Studies on the function and expression regulation of stress-responsive genes are dominant, but the link between stress-resistance-related signaling pathways and the mechanism of the entire signaling network system remains to be further studied. Although many research institutes have obtained various types of transgenic plants with certain resistance to salt and drought, through modern biotechnology, they have not yet reached the standard of industrialization. Therefore, there is still much work to be done to improve plant stress resistance.

SUMMARY OF THE INVENTION The present inventors cloned a coding gene of A. platensis monooxygenase (herein named CMO) by using SSH (suppression subtractive hybridization) and RACE (rapid amplification of cDNA ends), and determined its DNA sequence. Moreover, it was found that the transgenic plants were significantly improved in salt tolerance and drought tolerance after being introduced into plants by transgenic technology, and these traits were stably inherited.

 In a first aspect of the invention, a gene encoding a white bone monooxygenase CM0 (designated herein as AmCMO) having the sequence SEQ ID NO: 2 is provided.

A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, and the nucleotide sequence of the gene The expression control sequence of the recombinant expression vector is operably linked; preferably, the expression vector is pCAMBIA2300 _; preferably, the recombinant expression vector is the 35S CMO-2300 vector shown in Figure 2.

 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.

A fourth aspect of the present invention provides a method for improving salt tolerance and/or drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue And expressing the gene; preferably, the plant is Arabidopsis thaliana. A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Preferably, the plant is Arabidopsis thaliana.

 The sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving salt tolerance and/or drought tolerance of the plant And for use in plant breeding; preferably, the plant is Arabidopsis thaliana.

 The seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is set forth in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

 Figure l3⁄44 Construction process of the plant expression vector (35S-CMO-2300) of the CMO gene (Fig. la-lb). Figure 23⁄44 Plasmid map of the plant expression vector (35S CMO-2300) of the CMO gene.

 Fig. 3 shows the results of salt tolerance test of T1 Arabidopsis thaliana plants transfected with the AmCMO gene, and T113 showed significant salt tolerance, and the results of T1117 and T1119 were similar thereto, and are not shown here.

 Figure 4 shows the results of molecular level detection of the transcription level of the AmCMO gene in T1 transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. M is DNA Ladder Marker (DL2000), 1-8 is salt-tolerant T1 transgenic Arabidopsis plants (three strains T113, T1117, T1119, respectively), and 9-12 are non-transgenic control Arabidopsis plants.

 Fig. 5 shows the results of drought tolerance test of T1 Arabidopsis thaliana plants transfected with AmCMO gene, and T113 showed significant drought tolerance, and the results of T1117 and T1119 were similar thereto, and are not shown here.

BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

The unreported restriction endonucleases mentioned in the examples below were purchased from New England Biolabs. In the present invention, the proportions and percentages are calculated on a weight basis if not stated and not ambiguous in the context. Example 1. Construction of SSH library of Avicennia marina under salt stress: The specific method is:

A method according to PCR-select ^TM cDNA Clontech kit Subtraction Kit's instructions shown by suppression subtractive hybridization libraries constructed SSH (suppression subtractive library). During the experiment, the mRNA extracted from the root of A. serrata was used as a sample (Tester), and the mRNA extracted from the untreated Avicennia marina was used as a control. Specific steps are as follows:

 (1) Test materials:

 Avicennia is collected from Futian National Nature Reserve, Shenzhen, Guangdong Province (Ν22 ° 53 ', E114 ° 01 '). The hypocotyls of Aiic ni i maiina, which are not pest-resistant, well-developed and close to maturity, were collected, and hypocotyls of similar size, length and weight were selected for experiments. In plastic buckets (bottle diameter 18 cm, height 15 cm), the bottom of each bucket is padded with plastic trays, the fine sand is river sand, the average particle size is about 1 mm, tap water is washed, and each small barrel is planted with hypocotyls. 4. During 28 °C, natural light for 12 hours per day, during the long seedling culture period, a proper amount of tap water is added every day to replenish water, and Hoagland nutrient solution is poured once a week (DR Hoagland and DI Arnon. The water-culture method of growing plants Without soi l. Cal if. Agr. Expt. Sta. Circ. 347. 1950).

 (2) Material handling:

 The A. glabra seedlings with the same growth and development appearance (the seedling height is consistent and each seedling grows to 6 leaves) were divided into two groups. One group was poured with 2L of 500 mM NaCl, and one group was poured with 2 L of distilled water for 6 hours. Collect the roots of the treatment and control groups. After rapid freezing with liquid nitrogen, it was stored in a -70 °C refrigerator.

 (3) Total RNA extraction:

 Take 3.0 g of Avicennia leaves from the control and salt treatment groups, respectively, using the plant RNA extraction kit (purchased from

Invitrogen) extracts total RNA. The absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD ₂₆₀ / OD ₂₈₀ ratio was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose was used to coagulate. Gel electrophoresis detected the integrity of total RNA. The 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).

 (4) Suppression of subtractive hybridization:

The method according to Clontech's PCR-select ^TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization. Driver mRNA and Tester mRNA were reverse transcribed separately (reverse transcription primers were provided as primers) to obtain double-stranded cDNA, and 2 μg of Tester cDNA and 2 μg of Driver cDNA were used as starting materials for subtractive hybridization. . Tester cDNA and Driver cDNA were used in Rsa I at 37 °C water bath After digestion for 1.5 hours, the digested Tester cDNA was split into two equal portions, and the different linkers were ligated, while the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. The products of the two first forward subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed genes were amplified by two inhibitory PCR amplifications (PCR). Before, the second forward subtractive hybridization product is end-filled).

 (5) Construction and preliminary screening, cloning and identification of subtractive libraries

The second inhibitory PCR amplification product of the second forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) according to the instructions of the pGEM-T Easy kit (purchased from Promega) The specific steps are linked to the pGEM-T Easy vector as follows: The following components are sequentially added to the 200 l PCR tube: Purified combined positive subtractive hybridization cDNA fragment second inhibitory PCR product 3 μ 1 , 2 X T4 DNA ligase buffer 5 μl, pGEM-T Easy vector 1 μl, Τ4 DNA ligase 1 μl, ligated overnight at 4 °C. Then 10 μ ΐ was added to the reaction product, and added to 100 μL E. coli JM109 competent cells (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and then 250 l LB liquid medium was added. (containing 1% tryptone (purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID) and 1% NaCl (purchased from Sinopharm)) and placed in a 37 ° C shaker at 225 rpm Incubate for 30 minutes, then inoculate 200 μl of the bacterial solution from 50 μg/ml ampicillin, 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-milk Glycoside), 24 g/mL IPTG (isopropyl-β-D-thiogalactopyranoside) LB (same as above) solid (1.5% agar, the same below) on the culture plate (X-gal and IPTG are purchased From TAKARA), incubated at 37 ° C for 18 hours. Count the clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 300 white colonies (number: Am-SL-001 to Am-SL-300). The white colonies picked were inoculated into LB liquid medium (same as above) containing 50 μg/ml ampicillin in 96-well cell culture plate (CORNING), and cultured at 37 ° C overnight to add glycerol to the final concentration of glycerol. 20% (by volume), then stored at -80 °C for later use. The nested PCR primers Primer 1 and Primer 2R (PCR-select ^TM cDNA Subtraction Kit from Clontech) were used to perform PCR amplification verification on the cultured bacteria, and 224 positive clones were obtained, and then all positive clones were sent. Yingjie Jieji (Shanghai) Trading Co., Ltd. was sequenced.

 (6) cDNA sequencing analysis of differential clones:

After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 189 Expressed Sequence Tags (ESTs) (Unigene) were obtained. Example 2 Cloning of ABCMO, a gene encoding the albino monooxygenase

 After the cloned clone No. Am-SL-125 in the identified SSH library was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the monooxygenase. Here, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 is named ^ CMO, and the corresponding protein is named CMO.

 SEQ ID No: 3:

 1 ACTGAATACG TGATTTGTCG GGATGAAGAA GGAGCGTTGT ATGCATTTCA CAATGTATGC

61 CGCCATCATG GATCTCTTCT TGCCTCTGGA ACAGGGAAAA AGTCTTGCTT TGTCTGTCCA

121 TATCATGGTT GGACATATGG AATGGATGGT GCACTTGTAA AGTCAACAAG ACTAACAGGA

181 ATCAAGAACT TCAAAGTGAA TGAAATGGGA CTTGTTCCAC TCAAAGTCGC TATTTGGGGC

241 CCATTCATAT TAATCAATTT AGAAGGGGGG ATTACGCCGA AGGAAGGTCT AGATACCAAG

301 GTCGTGGCCA ATGAATGGCT AGGTAGTGCA GCAGAAGAGC TGAGCCTTGG TGGGGTTGAT

361 ACGTCATTGG ATTATGTTTG CAGACGTATA TATACCCTTG AATGCAATTG GAAGGTATTC

421 TGTGACAACT ATTTAGATGG TGGTTATCAT GTGCCATATG CACATAAAGA TCTTGCATCT

481 GGTCTCCAGC TTGATTCGTA TTCTACGGAA ATGTATGAAA GAGTTAGCAT TCAAAGCTGT

541 GGTGGCAATA CCAAGTGCAG TGAAGGAGAA TTTGATAGAC TGGGCTCAAA AGCTTTATAT

601 GCATTTGTCT ACCCCAACTT CATGATCAAT CGATATGGAC CATGGATGGA TACAAATCTT

661 GT

 Cloning of AmCMO full-length coding gene

 Based on the sequence of SEQ ID No: 3 that has been obtained, the following two specific primers were designed as the 5'-end specific primer for 3' RACE.

AmCMO GSP 1: SEQ ID No: 4:

 TGTGACAACT ATTTAGATGG TGG

 AmCMO GSP2: SEQ ID No: 5:

 The GGTCTCCAGC TTGATTCGTA TTC test procedure was performed according to the kit instructions (3 ' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).

 The first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal primer AUAP (provided with the kit), and the cDNA obtained by reverse transcription of the mRNA extracted by the salt treatment group was used as a template. Specific steps are as follows:

50 μ 1 PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 4 and AUAP each 2.0 μ ΐ and 35 μ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes. The obtained PCR product was diluted 50-fold with double distilled water, and then 2.0 μM was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:

 50 yl PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 5 and P AUAP Each of 2.0 μ 1 and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 A band of about 600 bp in the second round of PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method is the same as above) The transformed bacterial solution was applied to LB solid medium containing 50 μl of ampicillin and 40 μg of mL X-gaK 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. . Using SEQ ID NO: 5 and the universal primer AUAP for PCR amplification, 10 positive clones were obtained, and 3 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and 3 of the cDNA of the gene was obtained. 'End.

 Based on the obtained ^wCMO gene fragment, the following three specific primers were designed as the 3'-end specific primer of 5 ' RACE.

 AmCMO GSP3: SEQ ID No: 6:

 ACCTAGCCAT TCATTGGCCA CGAC

 AmCMO GSP4: SEQ ID No: 7:

 ATTCCATATG TCCAACCATG ATA

 AmCMO GSP5 : SEQ ID No: 8:

 GCAAGAAGAG ATCCATGATG GCG The experimental procedure was performed according to the kit instructions (5 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).

 Using SEQ ID NO: 7 and the universal primer AAP (provided with the kit), the cDNA obtained by reverse transcription of mRNA extracted from A. glabra L. (reverse transcription primer SEQ ID NO: 6, dCTP plus tail) was used as a template. The first round of PCR amplification, the specific steps are as follows:

50 μ 1 PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 Ex Taq (purchased from TAKARA), 10 μM primer SEQ ID NO: 7 and P AAP each with 2.0 μl and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C Denaturation for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 The obtained PCR product was diluted 50 times with double distilled water, and 2.0 μ ΐ was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and the primer AUAP. The specific steps are as follows:

 50 yl PCR reaction system: 5 μ 1 lO X Ex Buffer 3 μ 1 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 μ 1 Ex Taq 10 μM primer SEQ ID NO: 8 and P AUAP Each of 2.0 μ 1 and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

A band of about 600 bp in the second round of PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the specific method is the same as above) The transformed bacterial solution was applied to LB solid medium containing 50 μl of ampicillin and 40 μg of mL X-gaK 24 g/mL IPTG for screening. 10 white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), and stored at -80 ° C. . Using SEQ ID NO: 8 and primer AUAP to verify the bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), 8 positive clones were obtained, and 3 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The 5' end of the cDNA of the gene was obtained. After the obtained 5 'RACE product clone was sequenced, it was spliced with the above 3' RACE product sequencing result and the SEQ ID No: 3 sequence to obtain an AmCMO full-length cDNA sequence of SEQ ID No _: 9.

 SEQ ID No: 9:

 1 TGGTATCAAC GCAGAGTACG CGGGAGGACG GTGGACTATC TTCCCCGCAA GCACTGTATC

61 AACGGCCATG GCAGGGACCA CAGTGCTGCA GAAGCTCGTC TTTTTCCACC ACATGCCGGA

121 ATTCCCACAA CCTCAACAGC CTCGTAACCA ATTCGCCAAG AGACCCATTA ATAATCAAGT

181 TCGTCCTTTC AGCGACCACA GCCTTTGCCA CAGTAATAAC GGCAACTCTC TTCCAGTTAC

241 ATCCCCGACA AGACAAGAAA CGCGCGACAG CCATGGAAAG GTCGAAAGAC TGATCAAAGA

301 ATTCGACCCC AAAATTCCAA TAGAAGAAGC GATTACCCCG CCAAGCTCCT GGTACTCCGA

361 CCCTGATTTT TATTCTCACG AGCTCAGTCG GGTCTTCTAC AGAGGATGGC AGCCCGTTGG

421 ATATGTCGAT CAGCTTAAAG AGCCACGTGA TTTTTTCACT GGCAGATTGG GAAGTACTGA

481 ATACGTGATT TGTCGGGATG AAGAAGGAGC GTTGTATGCA TTTCACAATG TATGCCGCCA

541 TCATGGATCT CTTCTTGCCT CTGGAACAGG GAAAAAGTCT TGCTTTGTCT GTCCATATCA

601 TGGTTGGACA TATGGAATGG ATGGTGCACT TGTAAAGTCA ACAAGACTAA CAGGAATCAA

661 GAACTTCAAA GTGAATGAAA TGGGACTTGT TCCACTCAAA GTCGCTATTT GGGGCCCATT

721 CATATTAATC AATTTAGAAG GGGGGATTAC GCCGAAGGAA GGTCTAGATA CCAAGGTCGT

781 GGCCAATGAA TGGCTAGGTA GTGCAGCAGA AGAGCTGAGC CTTGGTGGGG TTGATACGTC

« Δ 1 901 CAACTATTTA GATGGTGGTT ATCATGTGCC ATATGCACAT AAAGATCTTG CATCTGGTCT

961 CCAGCTTGAT TCGTATTCTA CGGAAATGTA TGAAAGAGTT AGCATTCAAA GCTGTGGTGG

1021 CAATACCAAG TGCAGTGAAG GAGAATTTGA TAGACTGGGC TCAAAAGCTT TATATGCATT

1081 TGTCTACCCC AACTTCATGA TCAATCGATA TGGACCATGG ATGGATACAA ATCTTGTACT

1141 GCCACTGGGA CCCCGGAAAT GCCAAGTGAT TTTCGATTAT TTTCTAGATG CTTCTCTCAA

1201 GGACAACAGC ACATTCATCG AGAAGAGTTT AGAAGACAGT GAGAGGGTGC AGGAGGAAGA

1261 CATTTGTCTA TGCAATGCCG TACAGCTAGG CATGGACTCC CCAGCATACA ACGCTGGCCG

1321 GTATAGTCCA ACCGTCGAGA TGGCCATGCA TCATTTCCAT TGCCTGCTGC ATCAAAATCT

1381 CAGCACCTGT AAAGGATTTC GTTGAAACCG AAACTCCCAC ATCATGGCCT CTGTTCTCAT

1441 TCTCGTTCAT TTATCCAAAA CATGGTTTAG GACCAATCAA AAACCTGTAA ATGTATACTG

1501 CTCAGGTTAC CATTTGTCGG AAGCCTCACG TTGCAGTTGC AGTTGCAGTT GCAATTTCTT

1561 CTAAAAAAAA AAAAAAAAAA AAA A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows:

 SEQ ID No: 10:

 ATGGCAGGGA CCACAGTGCT GCA SEQ ID No: 11:

 TCAACGAAAT CCTTTACAGG TGC

 The CMO full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.

 PCR was performed using TAKARA's PrimeSTAR HS DNA polymerase as a template for the reverse transcription of mRNA extracted from the roots of Avicennia marina. 50 l PCR reaction system: 10 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 10 and P SEQ ID NO: 11 each of 2.0 μ 1 and 30 μ 1 of double distilled water.

PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.

PCR amplification product plus A tail: 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and then the resulting precipitate was dissolved in 21 μM of double distilled water. Then, 2.5 μl lO X Ex Buffer 0.5 μl 5 mM dATP, 1.0 l Ex Taq was added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained 1300 bp DNA fragment was recovered (Omega recovery kit), and ligated into the pGEM T-easy vector to obtain the ^wCMO-pGEM plasmid, and then the ligated product was transformed into E. coli JM109 competent cells (method) As above), the transformed bacterial solution was applied to LB solid medium containing 50 g/mL ampicillin and 40 g/mL X-gaK 24 g/mL IPTG for screening. Randomly pick 10 white colonies to inoculate respectively ^ > right, ^ ≡ ^ - h τ τ3 TI + i£ ^ it nb , + stand: ΦΙ^卄t≡ 廿, , /ώ 廿,, /ώ έ夂 The concentration is 20% (volume ratio), and it is stored at -80 °C for use. Using SEQ ID NO: 10 and SEQ ID NO: 11 for bacterial liquid PCR amplification verification (reaction system and reaction conditions are the same as above), 9 positive clones were obtained, and 3 positive clones were selected and sent to Yingjiejie (Shanghai) Trade. Sequencing, the resulting sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.

 Amino acid sequence of CMO protein: SEQ ID NO: 1

 1 MAGTTVLQKL VFFHHMPEFP

 21 QPQQPRNQFA KRPI NQVRP

 41 FSDHSLCHSN NGNSLPVTSP

 61 TRQETRDSHG KVERLIKEFD

 81 PKIPIEEAIT PPSSWYSDPD

 101 FYSHELSRVF YRGWQPVGYV

 121 DQLKEPRDFF TGRLGSTEYV

 141 ICRDEEGALY AFHNVCRHHG

 161 SLLASGTGKK SCFVCPYHGW

 181 TYGMDGALVK STRLTGIKNF

 201 KVNEMGLVPL KVAIWGPFIL

 221 INLEGGITPK EGLDTKVVAN

 241 EWLGSAAEEL SLGGVDTSLD

 261 YVCRRIYTLE CNWKVFCDNY

 281 LDGGYHVPYA HKDLASGLQL

 301 DSYSTEMYER VSIQSCGGNT

 321 KCSEGEFDRL GSKALYAFVY

 341 PNFMINRYGP WMDTNLVLPL

 361 GPRKCQVIFD YFLDASLKDN

 381 STFIEKSLED SERVQEEDIC

 401 LCNAVQLGMD SPAYNAGRYS

 421 PTVEMAMHHF HCLLHQNLST

 441 CKGFR*

Nucleotide sequence of AmCMO gene SEQ ID NO: 2

1 ATGGCAGGGA CCACAGTGCT GCAGAAGCTC GTCTTTTTCC ACCACATGCC GGAATTCCCA

61 CAACCTCAAC AGCCTCGTAA CCAATTCGCC AAGAGACCCA TTAATAATCA AGTTCGTCCT

121 TTCAGCGACC ACAGCCTTTG CCACAGTAAT AACGGCAACT CTCTTCCAGT TACATCCCCG

181 ACAAGACAAG AAACGCGCGA CAGCCATGGA AAGGTCGAAA GACTGATCAA AGAATTCGAC

241 CCCAAAATTC CAATAGAAGA AGCGATTACC CCGCCAAGCT CCTGGTACTC CGACCCTGAT

301 TTTTATTCTC ACGAGCTCAG TCGGGTCTTC TACAGAGGAT GGCAGCCCGT TGGATATGTC 421 ATTTGTCGGG ATGAAGAAGG AGCGTTGTAT GCATTTCACA ATGTATGCCG CCATCATGGA

481 TCTCTTCTTG CCTCTGGAAC AGGGAAAAAG TCTTGCTTTG TCTGTCCATA TCATGGTTGG

541 ACATATGGAA TGGATGGTGC ACTTGTAAAG TCAACAAGAC TAACAGGAAT CAAGAACTTC

601 AAAGTGAATG AAATGGGACT TGTTCCACTC AAAGTCGCTA TTTGGGGCCC ATTCATATTA

661 ATCAATTTAG AAGGGGGGAT TACGCCGAAG GAAGGTCTAG ATACCAAGGT CGTGGCCAAT

721 GAATGGCTAG GTAGTGCAGC AGAAGAGCTG AGCCTTGGTG GGGTTGATAC GTCATTGGAT

781 TATGTTTGCA GACGTATATA TACCCTTGAA TGCAATTGGA AGGTATTCTG TGACAACTAT

841 TTAGATGGTG GTTATCATGT GCCATATGCA CATAAAGATC TTGCATCTGG TCTCCAGCTT

901 GATTCGTATT CTACGGAAAT GTATGAAAGA GTTAGCATTC AAAGCTGTGG TGGCAATACC

961 AAGTGCAGTG AAGGAGAATT TGATAGACTG GGCTCAAAAG CTTTATATGC ATTTGTCTAC

1021 CCCAACTTCA TGATCAATCG ATATGGACCA TGGATGGATA CAAATCTTGT ACTGCCACTG

1081 GGACCCCGGA AATGCCAAGT GATTTTCGAT TATTTTCTAG ATGCTTCTCT CAAGGACAAC

1141 AGCACATTCA TCGAGAAGAG TTTAGAAGAC AGTGAGAGGG TGCAGGAGGA AGACATTTGT

1201 CTATGCAATG CCGTACAGCT AGGCATGGAC TCCCCAGCAT ACAACGCTGG CCGGTATAGT

1261 CCAACCGTCG AGATGGCCAT GCATCATTTC CATTGCCTGC TGCATCAAAA TCTCAGCACC

1321 TGTAAAGGAT TTCGTTGA

 Example 3 Construction of AmCMO Gene Plant Expression Vector

 The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The 35S promoter and the Tnos terminator were selected as promoters and terminators of the ^CMO gene, respectively. The construction flow chart is shown in Figure 1.

 Using primers SEQ ID NO: 12 and SEQ ID NO: 13, Pnos was amplified using the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TAKARA's PrimeSTAR HS DNA polymerase. 50 l PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 12 and SEQ ID NO : 13 each of 2.0 μ ΐ and 31 μ ΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with EcoRI, Bglll, and ligated into pCAMBIA2300 according to the kit instructions (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.

 SEQ ID NO: 12

 GCACGAATTC ggcgggaaac gacaatctga

 SEQ ID NO: 13

ATCCAGATCTAGATCCGGTGCAGATTATTTG Tnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 with the pBI121 plasmid as a template, using TAKARA's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ρΒΙ121 plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 14 and P SEQ ID NO: 15 each of 2.0 μ 1 and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was ligated by Kpnl, EcoRI digestion (Promega T4 ligase kit) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.

SEQ ID NO: 14:

 AAGGGTACCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 15:

 TCAGAATTCCCAGTGAATTCCCGATCTAGTA The 35S promoter was amplified using the primers SEQ ID NO: 16 and SEQ ID NO: 17 using the pCAMBIA2300 plasmid as a template. TAKARA's PrimeSTAR HS DNA polymerase was used. 50 yl PCR reaction system: 10 μ 1 5 X PS Buffer 3 μ 1 2.5 mM dNTP, 1.0 μl pCAMBIA2300 plasmid, 1.0 μl PrimeSTAR HS DNA polymerase, 10 μM primer SEQ ID NO: 16 and P SEQ ID NO: 17 each

2.0 μ ΐ and 31 μ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds), 72 ° C for 10 minutes. The resulting PCR product was ligated by HindIII and Sail (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3. SEQ ID NO: 16:

 ACTAAGCTTTAGAGCAGCTTGCCAACATGGTG SEQ ID NO: 17:

TGAGTCGACAGAGATAGATTTGTAGAGAGAGACT The full length sequence of the CMO-encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive ^wCMO-pGEM plasmid obtained in Example 2), using TAKARA's PrimeSTAR HS DNA polymerase. 50 μ ΐ PCR reaction system: 10 l 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 ^ CMO-pGEM plasmid, 1.0 μ 1 PrimeSTAR HS DNA polymerase, 10 μ Μ primer SEQ ID NO: 18 And SEQ ID NO: 19 each of 2.0 μΐ and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), 72 ° C extension Stretch for 10 minutes. The resulting PCR product was ligated by Sall and Kpnl (connection method as above) to pCAMBIA2300-3, and the plant expression vector 35S-CMO-2300 was obtained after verification (Fig. 2).

SEQ ID NO: 18

 ACTGTCGAC ATGGCAGGGA CCACAGTGCT GCA SEQ ID NO: 19

 GCTGGTACC TCAACGAAAT CCTTTACAGG TGC Example 4 35 S-AmCMO-2300 Expression Vector Transformation Agrobacterium

Preparation of competent cells of Agrobacterium tumefaciens GV3101 (purchased from Shanghai Maiqi Biotechnology Co., Ltd.): Agrobacterium GV3101 was inoculated on a solid medium containing 50 g/ml rifampicin and 50 g/m gentamicin LB. , culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 μ _§ /ιη1 galectin, and cultured overnight (about 12-16 hours) to OD ₆ at 28 °C. . . A value of 0.4 forms a seed broth. 5 ml of culture-activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μ _§ /ιη1 rifampicin and 50 g/ml gentamicin, and shake culture at 28 °C. -2.5 hours to OD ₆ . . =0.8. The ice bath solution was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state uniformly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; resuspend the cells by adding 1 ml of ice pre-cooled 10% (by volume) glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; 10% (by volume) of the pre-cooled glycerin was washed 3-4 times; then, the appropriate amount of ice pre-cooled 10% (volume ratio) of glycerol was used to resuspend the bacterial pellet to produce GV3101 competent cells at 40 μΐ/tube. It is packaged and stored at -70 °C for later use.

Transformation of Agrobacterium: The GV3101 competent cells were thawed on ice, and 1 μM of the plasmid 35S CMO-2300 obtained in Example 3 was added to 40 μM of the competent cells, and the mixture was mixed and ice-cooled for about 10 minutes. The mixture of the competent cells after the ice bath and the 35S-^1⁄27CMO-2300 plasmid was transferred to an ice-cold 0.1 cm size electric shock cup (purchased from Bio-Rad) using a micropipette, and the suspension was tapped to reach the suspension. The bottom of the electric shock cup (be careful not to have air bubbles). Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber. Set the program of MicroPulser (purchased from Bio-Rad) to "Agr" and apply an electric shock once. Immediately remove the electric shock cup and add 200 μΙ Β 预 medium pre-warmed at 28 °C. Quickly and gently mix the competent cells with a micropipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 1 hour at 225 rpm. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 g/ml rifampicin, 50 μ _§ /ιη1 gentamicin, 50 μ _§ /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Receptor Material Arabidopsis Culture

The rock with good water absorption and soft soil was mixed with nutrient soil (1:1) as the soil for Arabidopsis thaliana planting. Using a 9 cm diameter pot, seeded 20-30 Arabidopsis seeds per pot (Columbia type, from the Arabidopsis Bioresources Center, Ohio State University). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22V, light intensity 3500-4000 lx, photoperiod of 12 hours dark / 12 hours light culture, 1/2 MS liquid medium (9.39 mM KNO ₃ , 0.625 mM KH2PO ₄ , 10.3 mM NH ₄ NO _{3 )} every 7 days , 0.75 mM MgSO ₄ , 1.5 mM CaCl ₂ , 50 μ M KI, 100 μM MH ₃ BO ₃ , 100 μM MnSO ₄ - 30 u MZnSO ₄ - 1 μ MNa ₂ MoO ₄ - 0.1 μ M CoCl ₂ , 100 μ M Na ₂ EDTA, 100u M FeSO ₄ ). After 30 days of culture, 4-5 plants were kept per pot, and the photoperiod was adjusted to 8 hours dark/16 hours light culture. After most of the plants were bolted, the entire main moss was cut off at the base of the inflorescence, and the top edge was approximated. After 1 week, 4-6 new side mosses grow in the axillary bud, and when the flower buds form part of the flower buds and partially flower or form 1-2 pods, they can be used for transformation. Example 6 Arabidopsis flower leaching transformation

The GV3101 Agrobacterium liquid of the transformed 35S CMO-2300 expression vector obtained in Example 4 was inoculated to an LB liquid medium containing 50 μ _§ /ιη1 kanamycin (kan), and the next morning, press 1: 50 Inoculate into a new LB medium (1 L) containing 50 μ _§ /ιη1 kanamycin, and culture for about 8 hours to Agrobacterium OD ₆ . . Between 1.0 and 1.2. Centrifuge at 5000 rpm for 5 minutes at room temperature, discard the supernatant, and suspend the Agrobacterium pellet in the impregnation medium (1/2MS liquid medium and contain 5% sucrose; adjust to pH 5.7 with KOH; 0.02% Silwet L-77) , make OD ₆ . . At around 0.8. The upper part of the Arabidopsis thaliana prepared for transformation prepared in Example 5 was slowly and spirally immersed in the Agrobacterium-containing dyeing medium, and gently shaken clockwise for about 2 minutes, and covered with a transparent plastic cover. Humidity, put in the greenhouse overnight. After 24 hours, remove the plastic transparent cover and pour through the water. After 2-3 weeks, ensure that the plants are hydrated. When the plant stops flowering and the first fruit pod matures and turns yellow, it is covered with a paper bag. When all the pods in the paper bag turn yellow, the watering is stopped. After 1-2 weeks of drying, the seeds are collected and the transformants are screened. Untransformed Arabidopsis fruit pods were taken as controls. Example 7 Screening of transgenic positive transformants from Arabidopsis thaliana

Seed disinfection: Soak for 10 minutes with 70% ethanol, and occasionally suspend the seeds; then wash with sterile water four times, and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on a kanamycin containing 50 ug/ml. On the surface of 1/2MS solid screening medium (a maximum of 1500 seeds per 150 mm diameter plate), vernalized at 4 °C for 2 days, then at a constant temperature of 22 ° C, light intensity of 3500-4000 k, and a light cycle of 12 hours Incubate for 7-10 days in dark/12 hours light. After germination of the transgenic seeds on the screening medium for 2 weeks, the plants capable of germination and normal growth were transferred to soil for further cultivation. 1-2 leaves of each plant capable of normal growth on the screening medium were extracted, DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and conditions are the same as above) ), the PCR-negative plants were removed, and the seeds of the PCR-positive plants were collected and numbered (T011-T0123) and stored. Example 8 Planting of transgenic Arabidopsis thaliana T1 plants overexpressing CMO

 Select the vermiculite with good water absorption and soft soil to match the nutrient soil (1:1) as the soil for Arabidopsis planting. Will number

Each transformant of T011-T0123 and non-transgenic control Arabidopsis seeds were sown in 2 pots (20-30 seeds per pot). After sowing, the film is covered with a film to provide a moist environment for plant growth. Constant temperature 22 ° C, light intensity 3500-4000 lx, photoperiod of 12 hours dark / 12 hours light culture, 1/2 MS liquid medium per 7 days. After culturing for 25 days, 1-2 leaves were cut per plant and DNA was extracted as a template, and PCR was carried out using SEQ ID NO: 18 and SEQ ID NO: 19 as primers (reaction system and conditions are the same as above). PCR-negative plants were removed, and 7-8 PCR-positive vaccines were retained in each pot. After 10 days of culture, 5-7 transgenic Arabidopsis thaliana or non-transgenic control Arabidopsis thaliana seedlings with uniform size were kept in each pot for salt tolerance experiments. Example 9 Salt-tolerant experiment of transgenic Arabidopsis T1 plants overexpressing ^CMO

 In Example 8, the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana each retained a pot of plants for no treatment, normal watering

1/2MS liquid medium, one pot of each plant was irrigated with 1/2MS liquid medium containing 150 mM NaCl, constant temperature 22 ° C, light intensity 3500-4000 k, 12 hours light culture / 12 hours dark culture cycle, 14 The results of the experiment were observed after the day. The salt tolerance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the T1 transgenic plants T113, T1117 and T1119 showed significant salt tolerance (see Figure 3, with T113). For example, the results of T1117 and T1119 are similar, not shown here). Example 10 Verification of the expression of the CMO gene at the transcriptional level

Eight T1 transgenic plants with good salt tolerance in Example 9 were randomly selected (one of the above-mentioned T113, T1117, T1119 three salt-tolerant strains), and the control plants in Example 9 were randomly selected from 4 plants. The leaves (0.05 mM) were treated with salt (150 mM NaCl) for 14 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA obtained at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay [J box Superscript III Reverse Transcriptase, and 1 total RNA was used as a template for reverse transcription. The CMO fragment was amplified using primers SEQ ID NO: 10 and SEQ ID NO: 20 (SEQ ID NO: 20: GGCACATGAT AACCACCATC TAA), and its transcription was examined. The AtACT2 fragment was amplified using the SEQ ID NO: 21 (SEQ ID NO: 21: 5-GCCATCCAAGCTGTTCTCTC-3) and SEQ ID NO: 22 (SEQ ID NO: 22: TTCTCGATGGAAGAGCTGGT) (Arabidopsis housekeeping gene: Http:〃 www.ncbi.nlm. nih.gov/nuccore/AK317453.1 ) , as a photo.

 The PCR reaction was carried out using the Ex DNA polymerase of TAKARA and using the cDNA obtained by the above reverse transcription as a template. 50 μl ΡΟ Reaction system: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 0.3 μΐ Ex DNA polymerase, 10 μΜ primer SEQ ID NO: 10 and P SEQ ID NO: 20 each 2.0 μ1, and 35.7 ΐ ΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 30 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.

 The electrophoresis results of the PCR products are shown in Figure 4: M is the DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), and 1-8 is the salt-tolerant T1 transgenic Arabidopsis plants (T113, Τ1117, T1119, respectively). Three strains), 9-12 are non-transgenic control Arabidopsis plants. The results showed that there was significant transcription of ^wCMO in the salt-tolerant T1 transgenic Arabidopsis plants, and there was no transcription of ^wCMO in the non-transgenic control Arabidopsis plants. Example 11 Drought tolerance experiment of T113, T1117, T1119 transgenic Arabidopsis thaliana

 T113, T1117, T1119 transgenic Arabidopsis thaliana were planted in the same manner as in Example 8. Transgenic Arabidopsis thaliana and control Arabidopsis thaliana were treated without treatment. Normally watered 1/2MS, transgenic Arabidopsis thaliana, another pot of Arabidopsis thaliana , do not water 1/2MS, constant temperature 22 ° C, light intensity 3500-4000 k, 12 hours light culture / 12 hours dark culture cycle. The results of the experiment were observed after 10 days: Tlj2 showed significant drought tolerance (see Fig. 6, in T113 case, and the results of T117 and T1119 are similar thereto, not shown here).

Claims

Claim A protein encoding a gene encoding a monooxygenase of A. glabra, having the sequence of SEQ ID NO: 1.  2. A gene encoding the protein of claim 1, the sequence of which is SEQ ID NO: 2.  A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and wherein the nucleotide sequence of the gene and the expression control sequence of the expression vector are operably Preferably, the expression vector is pCAMBIA2300.  4. The recombinant expression vector of claim 3 which is the 35S CMO-2300 vector shown in Figure 2.  A recombinant cell comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.  A method for improving salt tolerance and/or drought tolerance of a plant, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and causing the gene Expression; Preferably, the plant is Arabidopsis thaliana.  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.  8. The method of claim 7, wherein the plant is Arabidopsis thaliana.  9. The gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 for improving salt tolerance and/or drought tolerance of a plant and for use in plant breeding .  10. The use of claim 9, wherein the plant is Arabidopsis thaliana.